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Successful Clinical Implementation of Corneal Epithelial
Stem Cell Therapy for Treatment of Unilateral Limbal
Stem Cell Deficiency
Sai Kolli, Sajjad Ahmad, Majlinda Lako, Francisco Figueiredo
TRANSLATIONAL AND CLINICAL RESEARCH
Successful Clinical Implementation of Corneal Epithelial Stem Cell
Therapy for Treatment of Unilateral Limbal Stem Cell Deficiency
SAI KOLLI,a,b,c SAJJAD AHMAD,a,b,c MAJLINDA LAKO,a,b FRANCISCO FIGUEIREDOa,b,c
a
North East Institute for Stem Cell Research and bInstitute of Human Genetics, University of Newcastle upon
Tyne, International Centre for Life, NE1 3BZ, United Kingdom; cDepartment of Ophthalmology,
Newcastle University, Royal Victoria Infirmary, Newcastle Upon Tyne, United Kingdom

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Key Words. Ex vivo expansion • Corneal epithelial stem cells • Limbal stem cells • Limbal stem cell deficiency

ABSTRACT
The corneal epithelium is maintained by a population of
stem cells known as limbal stem cells (LSCs) due to their
location in the basal layer of the outer border of the cor-
nea known as the limbus. Treatment of limbal stem cell
deficiency (LSCD) has been achieved with transplantation
of ex vivo expanded LSCs taken from a small biopsy of
limbus. This is a relatively new technique, and as such,
specific national or international guidance has yet to be
established. Because of the lack of such specific guidance,
our group has sought to minimize any risk to the patient
by adopting certain modifications to the research method-
ologies in use at present. These include the replacement of
all non-human animal products from the culture system
and the production of all reagents and cultures under
Good Manufacturing Practice conditions. In addition, for
the first time, a strictly defined uniform group of patients
with total unilateral LSCD and no other significant ocular
conditions has been used to allow the success or failure of
treating LSCD to be attributable directly to the proposed
Disclosure of potential conflicts of interest is found at the end of this article.
stem cell therapy. A prospectively designed study with
strict inclusion and exclusion criteria was used to enroll
patients from our database of patients with unilateral
LSCD. Eight eyes of eight consecutive patients with unilat-
eral total LSCD treated with ex vivo expanded autologous
LSC transplant on human amniotic membrane (HAM)
with a mean follow-up of 19 (RANGE) months were
included in the study. Postoperatively, satisfactory ocular
surface reconstruction with a stable corneal epithelium
was obtained in all eyes (100%). At last examination, best
corrected visual acuity improved in five eyes and remained
unchanged in three eyes. Vision impairment and pain
scores improved in all patients (p < .05). This study dem-
onstrates that transplantation of autologous limbal epithe-
lial stem cells cultured on HAM without the use of non-
human animal cells or products is a safe and effective
method of reconstructing the corneal surface and restoring
useful vision in patients with unilateral total LSCD. STEM
CELLS 2010;28:297–610

mammals [4]. The limbal epithelium acts as a reservoir for

INTRODUCTION
The cornea is the clear dome-shaped window at the front of
the eye, and its clarity and regular surface is vital for the
transmission and focusing of light onto the retina, allowing
accurate visual perception. Corneal disease represents the sec-
ond most common cause of world blindness after cataract [1].
The surface of the cornea is made up of an epithelium, which
is continuous with that of the surrounding conjunctiva. The
transition between the corneal and conjunctival epithelia is
formed by the limbal epithelium. There is now a substantial
body of evidence, both scientific and clinical, pointing to the
basal layer of the limbus epithelium as the location for puta-
tive corneal epithelial stem cells, CESCs, also known as lim-
bal stem cells, LSCs [2, 3], although recent findings suggest a
diffuse distribution of LSCs on the ocular surface in certain
the replacement of corneal epithelial cells that are normally
continually lost from the corneal surface into the tear film. In
addition, the limbal epithelium is thought to exert a ‘‘barrier’’
function in preventing the migration of conjunctival epithe-
lium and its blood vessels on to the surface of the cornea.
Upon significant injury to the limbal epithelium and the LCSs
contained therein, the corneal epithelium cannot renew itself
and conjunctival epithelium can encroach on to the corneal
surface, a process called ‘‘conjunctivalization’’. This results in
persistent epithelial defects and neovascularization of the cor-
nea; chronic inflammation; scarring, and loss of vision. This
is known clinically as limbal stem cell deficiency (LSCD).
Corneal vascularization and opacity have been estimated to
cause blindness in eight million people (10% of total blind-
ness) worldwide each year [1], and various forms of LSCD
contribute to this total. A large number of ocular surface

Author contributions: S.A. and S.K.: Conception and design of the study, collection and assembly of data, data analysis and
interpretation, manuscript writing and clinical work; S.A. and S.K. contributed equally to this work; M.L.: Conception and design of the
study, fund raising, data analysis and interpretation, and manuscript writing; F.C.F.: Conception and design of the study, fund raising,
collection and assembly of data, data analysis and interpretation, manuscript writing, and clinical work.
Correspondence: Majlinda Lako, PhD, Newcastle University, Institute of Human Genetics and NESCI, International Centre for Life,
Central Parkway, Newcastle upon Tyne NE1 3BZ, U.K. Telephone: 00 44 191 241 8688; Fax: 00 44 191 241 8,666; e-mail: Majlinda.
Lako@ncl.ac.uk Received September 24, 2009; accepted for publication December 1, 2009; first published online in STEM CELLS
EXPRESS December 10, 2009. V
C AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.276

STEM CELLS 2009;28:597–610 www.StemCells.com

598
diseases, both acquired and congenital, share features of par-
tial or complete LSCD [2]. This means that the diagnosis of
LSCD must be considered in all cases of significant corneal
epithelial disease and vascularization because only the
replacement of the LSC population will be effective in treat-
ing these conditions.
The management of LSCD depends on whether the patient
has partial or total LSCD. In partial LSCD, there is still lim-
ited presence of functioning LSCs, and when the visual axis
is covered with normal corneal epithelium and the patient is
relatively asymptomatic with good vision, conservative [medi-
cal] management is indicated [3]. However, in partial LSCD,
where there is central corneal involvement with decreased
vision, significant irritation, and persistent epithelial defect,
surgical management including mechanical debridement of
conjunctival epithelium from the corneal surface and/or amni-
otic membrane transplantation may be indicated [5]. However,
in total LSCD, where there is no evidence of functioning
LSCs, the only treatment is surgical and involves a stem cell
therapy that allows the replacement of the damaged or absent
LSC population.
In the past, central corneal transplants were used with lim-
ited long-term success to treat extensive LSCD [6–8]. The
main reason for failure is the inability to maintain a healthy
corneal epithelium, which relies solely upon a healthy re-
cipient limbus. In total LSCD, recipient limbal epithelium
has to be restored in the first instance, so that a healthy cor-
neal epithelium can be maintained. This can only be
achieved by the transplantation of healthy limbal epithe-
lium, using either large whole tissue limbal epithelial grafts
[9–13] or, more recently, ex vivo expanded limbal epithelial
graft from small biopsies of limbal epithelium, which can
be taken from patients’ other healthy eye (in the case of
unilateral LSCD: autograft) or healthy eye of living related
or cadaveric donors (in the case of bilateral LSCD: allo-
graft) [14]. The requirement of much smaller amounts of
tissue for the ex vivo expansion process means that patients
with unilateral LSCD have two main advantages. Firstly,
the small amounts of tissue required for ex vivo expansion
are much less likely to damage the LSC population of the
healthy donor eye than previously used autologous whole
tissue grafts that required large quantities of limbal tissue
for direct transplantation. Secondly, the donor tissue is au-
tologous and eliminates the requirement of systemic
immune suppression compared to previously used whole tis-
sue allografts. More recently, ex vivo expansion of oral mu-
cosa epithelium has been used successfully to transplant
onto the ocular surface of rabbits and subsequently in
humans with total bilateral LSCD, which will potentially
provide treatment for autologous stem cell therapies for
patients with total bilateral LSCD [15–18].
The ex vivo expansion of limbal epithelium prior to trans-
plantation is a relatively new technique, and as such, specific
national or international guidance has yet to be established,
although regulatory bodies encourage strategies that minimize
any risk to the patient [19]. Because of the lack of such spe-
cific guidance, our group has sought to minimize any risk to
the patient by adopting certain modifications to the research
methodologies in use at present. These include, firstly, the
replacement of all non-human animal products from the cul-
ture system and, secondly, the production of all reagents and
cultures under Good Manufacturing Practice (GMP) condi-
tions. These modifications have been used to produce ex
vivo expanded limbal epithelium to then treat a uniform
group of patients who all have total unilateral LSCD and no
other significant ocular conditions. This meant that all treat-
ments were autologous, requiring no immunosuppression;
Successful Stem Cell Therapy for Unilateral LSCD
thus, any success or failure of the stem cell therapy could be
attributed directly to our intervention and not due to the
effects of concomitant immunosuppression or ocular co-
morbidity.
Up to now, the initial growth of limbal epithelial cells in
culture required the concomitant use of non-human animal
cells and products including a mouse 3T3 fibroblast feeder
layer for co-culture and fetal calf serum (FCS) in the growth
medium, and this poses two problems: Firstly, since such a
transplant would be a potential xenograft, the patient may
require immunosuppression to prevent rejection of the tissue.
Secondly, and more importantly, the use of non-human animal
products in tissue destined for human transplantation has the
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potential to produce interspecies pathogen transfer [20, 21].
This latter risk would be augmented further on a background
of immunosuppression. The successful culture of LSCs has
been established on extracellular matrix components including
collagen IV coated shields [22], laminin and fibronectin [23],
human limbal fibroblasts [24], and human amniotic membrane
(HAM) [25–30]. In addition, defined serum-free media has
been successfully used for expansion of LSCs, although the
limbal cell proliferation was reduced in comparison to serum-
containing media [31]. Instead, human autologous serum
(HAS) has been successfully used to replace the need for FCS
in epithelial growth medium for the culture of a variety of
epithelial cell types including skin [32], oral mucosa [33], and
cornea [34].
In this study, we report for the first time successful ex
vivo expansion of limbal epithelial cells using a completely
non-human animal product free system that combines usage
of HAM as a matrix and HAS as a replacement for FCS in
culture medium. Such a system of ex vivo expansion was
fully validated and refined over a three-year period in the lab-
oratory prior to implementation in our first patients. The stem
cell therapy was fully studied and approved by all the relevant
ethical and regulatory bodies in the United Kingdom prior to
human translation. The ex vivo expanded autologous limbal
epithelium was then transplanted into eight patients with uni-
lateral total LSCD. Successful treatment of LSCD was proven
for each patient in this study both from reversal of the clinical
signs and also by the conversion of impression cytology
specimens of the central cornea from a conjunctival pheno-
type preoperatively to an exclusive corneal phenotype
postoperatively.
MATERIALS AND METHODS
Ethics and Regulatory Statements
The ex vivo expansion of autologous human limbal epithelium for
the treatment of LSCD was approved by the Human Tissue
Authority (HTA), Local Research Ethics Committee (LREC), and
Newcastle upon Tyne Hospitals NHS Foundation Trust’s Research
and Development Department and New Procedures Committee.
For more detail, see Supporting Information data, Annex 1.
Patient Demographics
Eight patients (that fulfilled all the inclusion and exclusion crite-
ria; Supporting Information data, Annex 1) were included in this
study. Detailed patient demographics are shown in Supporting In-
formation Table 1. Seven of the patients were male, and one was
female. The mean age was 43 (range 16-73). Mean follow-up
was 19 months (range 12-30). All patients had total unilateral
LSCD confirmed by both clinical examination and impression cy-
tology with cytokeratin profiling. Consent was obtained from
Kolli, Ahmad, Lako et al.
Patient 1 to provide clinical pictures for this manuscript. The con-
sent form is saved in the clinical notes.
Study Protocol
The key steps used for ex vivo expansion of limbal stem cells
and subsequent transplantation are outlined in Supporting Infor-
mation Figure 1. In summary, following diagnosis of LSCD, the
technique involved taking a small limbal biopsy from the contra-
lateral healthy eye of a patient with unilateral LSCD and expand-
ing the LSC population ex vivo and then transplanting this
enlarged population into the surgically prepared diseased fellow
eye.
Impression Cytology
All patients underwent objective assessment of corneal epithelium
in both eyes by impression cytology. This was performed to con-
firm the diagnosis of total LSCD in the affected eye and to con-
firm absence of any features of LSCD in the fellow healthy eye.
For more information on this technique, please refer to Support-
ing Information Figure 2 and Supporting Information data, Annex
1.
Manufacture of Human Autologous Serum
Following a negative infectious disease screen for hepatitis B and
C, HIV, HTLV, and syphilis, the patient was eligible to proceed
to have blood taken for HAS preparation/production. Although no
universally accepted system of HAS production exists, the tech-
nique we used is based on the optimized protocol [36]. HAS pro-
duced with this protocol has been used and validated in our pre-
ceding laboratory studies and shown to support limbal epithelium
expansion while maintaining LSC morphology, colony-forming
efficiency (CFE), and expression of putative LSC markers (data
not shown). All HAS production was carried out within the GMP
Stem Cell Laboratory using the protocol outlined in Supporting
Information Figure 3.
Plating of Human Amniotic Membrane
HAM for clinical transplantation was obtained from the NHSBT
Tissue Services (London & Liverpool, U.K.). For plating of
explants on HAM, please refer to Supporting Information data,
Annex 1.
Ex Vivo Expansion Protocol of LSCs on HAM
Limbal biopsy was performed as shown in Supporting Informa-
tion data, Annex 2, and Supporting Information Figures 3 and 4.
In brief, the epithelial medium from the previously plated HAM
was removed from the culture well. The limbal biopsy was
placed on the center of the plated HAM and gently pressed
downward to promote the attachment, and 1.5 ml of epithelial
medium was gently added to the culture well very slowly. The
culture was examined and subsequently fed with 2 ml of epithe-
lial medium on alternate days up to 12-14 days (Supporting Infor-
mation Fig. 5). The area of explant outgrowth was marked on the
underside of the culture well at the time of each feed to allow
subsequent measurement of growth rate (Supporting Information
Fig. 6). On the 7th–10th day of culture, 1 ml of the culture me-
dium was sent for microbiological analysis. Early explant out-
growth was observed between days 2 and 3; full coverage of the
extent of the HAM was usually reached between 12 and 14 days.
For control purposes, ex vivo expansion of LSCs using 3T3
feeder cells and fetal calf serum as described in [37] was carried
out simultaneously in the initial experiments (data not shown).
Transplantation Protocol
Transplantation of the ex vivo expanded limbal tissue on to
HAM took place between 12 and 14 days following the initial
limbal biopsy. Full informed consent was obtained from all
patients prior to surgical intervention. For full details of the pro-
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599
tocol, refer to Supporting Information data, Annex 3, and Sup-
porting Information Figure 7.
Follow-up Protocol
Postoperatively, patients were treated with a combination of pre-
servative-free 0.5% chloramphenicol antibiotic eye drops (Min-
ims, Chauvin, U.K.), 1% prednisolone acetate steroid eye drops
(Moorfields Eye Hospital, London), and autologous serum (pro-
duced from the patient’s own blood by NHSBT, U.K.). The
patients were reviewed at day 1, week 1, week 2, month 1, month
2, and month 3 postoperatively and then at least at 3 monthly
intervals in addition to extra visits dictated by clinical need. Clin-
ical examination was routinely carried out independently by two
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authors (S.K. and F.F.), and clinical photographs were taken at
each 3-month visit. To ensure minimal disturbance to the trans-
planted epithelium, impression cytology was performed only at
6 months post-transplantation and 6 monthly thereafter. If the
LSCD had been successfully reversed at 12 months post LSC
transplantation, the patient would be considered if needed for
subsequent penetrating keratoplasty depending on the degree of
previous corneal scarring and consequent best corrected visual
acuity.
Clinical Outcome Assessment
At each 3 monthly visit, subjective pain and vision scores were
measured using the same assessment score cards as used preoper-
atively. The patient was independently examined by two authors
(S.K. and F.F.), and the clinical features related to LSCD were
noted with particular reference to all the initially defined objec-
tive outcomes including presence of epithelial defect, presence of
active corneal vascularization, and corneal opacity. In addition,
Snellen’s chart best corrected and LogMAR visual acuity was
assessed by at least two independent assessors at each visit. Cor-
neal impression cytology was performed at 6 monthly intervals.
Colony Forming Efficiency Assays
In order to determine the efficiency of the epithelial cells to form
colonies under different growth conditions, colony forming effi-
ciency (CFE) assays were performed. For more detail, please
refer to Supporting Information data, Annex 1.
Statistical Analysis
For two groups of data, the student’s paired t-test was used to
obtain probability (p) values. For three or more groups of univari-
ate data, single-factor analysis of variation (ANOVA) was used
to obtain p values. For three or more groups of bivariate data
(with replicates), two-factor ANOVA with replication was used
to obtain p values. Results with p values of less than 0.05 were
considered statistically significant.
RESULTS
Successful Laboratory Ex Vivo Expansion of
Limbal Epithelial Cells Using a Combination
of HAM and HAS
Human limbal epithelial cells were successfully cultured on
HAM as both explant (Fig. 1A and 1B) and single-cell sus-
pension cultures (Fig. 1C and 1D) in fetal calf serum (FCS)
containing media. The outgrowth areas were measured at
weekly intervals, and this indicated that both the explant (Fig.
1E; p < .00005; n ¼ 3) and suspension cultures showed suc-
cessful increase in growth (data not shown). Comparison
between the two culture types indicated that the limbal
explant culture epithelial outgrowth covered the amniotic
membrane much earlier than the suspension cultures (Fig. 1F;
p < .005; n ¼ 3). We investigated whether the ex vivo
600 Successful Stem Cell Therapy for Unilateral LSCD

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Figure 1. Human limbal explant and cell suspension cultures on human amniotic membrane. (A): Macroscopic picture of culture showing two
inner red rings indicating previous day’s growths and the present outgrowth as the outer ring. (B): Phase contrast micrograph of the culture show-
ing the explant in the upper right corner and the epithelial outgrowth on the amniotic membrane. Scale bar ¼ 200 lm. (C): Macroscopic picture
of culture showing multiple limbal epithelial colonies highlighted by the black asterisks. (D): Phase contrast micrograph of the culture showing
two limbal epithelial colonies as highlighted by the white arrows. The 200 lm scale bar is shown. (E): Epithelial outgrowth areas from limbal
explants on amniotic membrane with increasing time in culture. The number of days is shown on the x-axis, and the epithelial outgrowth areas
from the human limbal explants are shown on the y-axis. (F): The number of days for the cultured epithelial cells to cover the amniotic mem-
brane in both limbal explant and cell suspension cultures. The type of culture (explant or cell suspension) on amniotic membrane is shown on
the x-axis, and the number of days to cover the entire amniotic membrane is shown on the y-axis (* indicates p < .005; n ¼ 3).

single-cell expansion of limbal epithelial cultures could be
achieved on 3T3 feeders using medium supplemented with
HAS as a replacement for FCS. Morphological observations
showed that the HAS cultures were identical to co-cultures
using FCS (Fig. 2A). Cell counts performed on both the FCS
and HAS containing primary cultures showed that those with
HAS had significantly higher cell counts (Fig. 2B; p < .05; n
¼ 3). However, there was no statistically significant differ-
ence (n ¼ 3) between the colony-forming efficiency (CFE) of
the limbal epithelial cells from cultures established using ei-
ther FCS or HAS (Fig. 2C) or p63 expression (Fig. 2D).
For clinical applications, we combined the explant culture
method on HAM with medium supplemented with HAS. Mor-
phological observations, flow cytometry analysis for expres-
sion of p63, and CFE assays indicated that there were no stat-
istically significant differences between the two culture
methods (data not shown). A comparison of the outgrowth
areas from explant on HAM using FCS and HAS (Fig. 2E)
showed that the HAS-containing cultures had significantly
larger outgrowths (p < .005; n ¼ 3). This was corroborated
by higher cell counts in the HAS ex vivo expanded cultures
(data not shown).
Prior to carrying out cultures of limbal explants for
patients with total unilateral LSCD, a series of cultures using
cadaveric human limbal explants were first established in the
GMP Stem Cell Laboratory. The three trial cultures of 1.5
mm by 1.5 mm cadaveric human limbal explants on HAM
using human serum containing epithelial medium grew
Kolli, Ahmad, Lako et al. 601

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Figure 2. Comparison of human autologous serum to fetal calf serum. (A): Phase contrast micrograph of human limbal epithelial cells co-cul-
tured with 3T3 fibroblasts using human serum containing medium. Early epithelial colonies resembling holoclone-like colonies can be seen high-
lighted by the white arrows. The 200 lm scale bar is shown. (B): Total viable cell counts of human limbal epithelial cells co-cultured with 3T3
fibroblasts using fetal calf serum or human serum in the growth medium. The different culture conditions (fetal calf serum or human autologous
serum) are shown on the x-axis, and the total cell count is shown on the y-axis (* indicates p < .05; n ¼ 3). (C): Colony forming efficiencies of
human limbal epithelial cells co-cultured with 3T3 fibroblasts using fetal calf serum or human serum in the growth medium. The different culture
conditions (fetal calf serum or human serum) are shown on the x-axis, and the colony-forming efficiency is shown on the y-axis. (D): Colony-
forming efficiencies of human limbal epithelial cells co-cultured with 3T3 fibroblasts using fetal calf serum or human serum in the growth me-
dium. The different culture conditions (fetal calf serum or human serum) are shown on the x-axis, and the colony forming efficiency is shown on
the y-axis. (E): Explant outgrowths from limbal explants on human amniotic membrane using fetal calf serum and human serum. The outgrowth
areas at weekly intervals were measured. The day of outgrowth is shown on the x-axis, and the outgrowth area is shown on the y-axis.

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602 Successful Stem Cell Therapy for Unilateral LSCD

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Figure 3. Preoperative assessment of Patient 1. (A): Preoperative clinical photograph of left eye with total limbal stem cell deficiency (LSCD)
showing complete corneal vascularization and significant corneal opacification. (B): Fluorescein staining of the same eye observed with cobalt
blue filter reveals an irregular epithelial surface with diffuse epithelial late staining. (C): The fellow right eye is unaffected with a normal corneal
examination. (D): Normal corneal impression cytology with minimal cellularity with firm impression and no CK19 positive cells. (E): Impression
cytology showing extensive cellularity and CK19 positive staining diagnostic of LSCD.

significantly better under GMP conditions than in the standard
tissue culture laboratory (Supporting Information Fig. 8; p <
.00005; n ¼ 3).
Successful Transplantation of Ex Vivo Expanded
Limbal Epithelial Cells into Patient with LSCD:
First Case Study
The first transplantation of autologous ex vivo expanded lim-
bal epithelial cells under GMP conditions was performed on a
45-year-old Caucasian male who had developed all the clini-
cal features of total LSCD (following a severe chemical injury
to the eye) including (i) significant epithelial defect; (ii) sig-
nificant peripheral and central corneal vascularization; (iii)
marked corneal opacity [Grade 4, no iris details visible]; and
(iv) total loss of Palisades of Vogt [Fig. 3A and 3B; for a full
clinical history, see Supporting Information data, Annex 4].
The right eye examination was entirely normal (Fig. 3C). The
patient’s subjective pain score was 5/10 and the visual impair-
ment score was 7/10 with regards to the left eye (using the
scale provided in Supporting Information Figure 9) and 0/10
in either case for the normal right eye. Impression cytology of
both central corneas confirmed left total LSCD and normal
right corneal epithelium (Fig. 3D and 3E).
Right limbal biopsy was obtained from the patient as pre-
viously described in Supporting Information data, Annex 2. A
sheet of ex vivo expanded epithelium was established at 14
days after initial biopsy. On day 14, the patient underwent
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Figure 4. Superficial keratectomy specimen at time of limbal stem cell transplantation surgery. (A): Clinical photograph of the left eye of
Patient 1 showing the removal of abnormal epithelium from the surface of the left eye with total limbal stem cell deficiency. (B): Low-power his-
tology of the excised central conjunctivalized corneal epithelium stained with hematoxylin and eosin (H&E) showing a very abnormal multilay-
ered epithelium, which contains multiple blood vessels (arrows) that are absent in normal cornea; scale bar ¼ 500 lm. (C): Higher-power H&E
stained excised epithelium showing in more detail a very thickened epithelium with irregular surface and loose sloughing epithelium with multi-
ple goblet cells (arrows). (D): PAS-stained excised epithelium clearly demonstrating the glycogen-containing goblet cells (arrows). (E): Immuno-
histochemistry of the excised epithelium demonstrates the lack of the corneal specific marker cytokeratin K3 and (F) the heavy expression of the
conjunctival marker, CK19. Scale bars for (C–F) ¼ 100 lm.

total superficial keratectomy to remove all the abnormal cor- tissue was highly vascular, irregularly arranged with multiple
neal epithelium from his left LSCD eye. Histological and goblet cells and with strong expression of CK19 and lack of
immunohistochemistry analysis confirmed that the removed CK3 expression confirming complete absence of a corneal
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604 Successful Stem Cell Therapy for Unilateral LSCD

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Figure 5. Analysis of ex vivo expanded epithelium of Patient 1. (A): Summary panel showing the light microscopic and immunohistochemistry
appearances for ex vivo expanded epithelium of Patient 1. H&E staining reveals an epithelium with a basal layer of tightly packed cuboidal cells
that express high levels of p63, ABCG2, Vimentin, & Ki67. Conversely, the basal layer is devoid of high CK3 expression. (B): TEM of cultured
epithelium from Patient 1. [a] The ex vivo cultured epithelium (EE) sits on the HAM. The basal cells are much smaller and cuboidal (red outline)
with high N/C ratios (blue outline) compared with the large columnar cells of adult corneal epithelium. Areas of the expanded epithelium (shown
by the dotted rectangles) were viewed at higher magnifications to reveal [b] the presence of MP on the superficial epithelial cells, [c] the pres-
ence of desmosomes connecting the basal cells together, and [d] hemidesmosomes connecting the basal cells to the basement membrane. Abbre-
viations: HAM, human amniotic membrane; MP, microplicae.

epithelium phenotype (Fig. 4). The ex vivo expanded limbal
epithelium sheet on HAM was secured in place (stromal side
down) with four 10-0 nylon sutures. Also, the explant was
positioned in the limbus at 12 o’clock to allow the highest
concentration of LSCs to be placed in the natural protective
niche environment [38]. This was followed by placement of a
second HAM on top of the first HAM containing the LSC
explant (stromal side down). At the time of surgery, excess
cultured limbal epithelium on HAM was excised and divided
into two halves: one for histology and immunohistochemical
analysis and the second for ultrastructural analysis of the cul-
tured epithelium by transmission electron microscopy (TEM).
This analysis confirmed that a normal limbal epithelium is
formed on the HAM (Fig. 5A). This epithelium is 2-3 cell
layers thick, although it is thicker at the edges of the culture
specimen. The basal layer of cells adjacent to the HAM
shows characteristics indicating an actively expanding LSC
phenotype including (i) small cuboidal cells with undifferenti-
ated appearance; (ii) high expression of the putative LSC
markers p63, ABCG2, and vimentin; (iii) low expression of
the CK3 differentiation marker; and (iv) high expression of
the cell proliferation marker Ki67. TEM confirmed the light
microscopy findings demonstrating the formation of a primi-
tive epithelium with a prominent basal layer of cuboidal cells
Kolli, Ahmad, Lako et al.
Figure 5.
with a high nucleus/cytoplasm ratio (Fig. 5B). The basal cells
are attached to their basement membrane via hemidesmo-
somes and to each other via desmosomes. The superficial
squamous cells, which are away from the HAM, showed early
differentiation demonstrating primitive microplicae formation
in keeping with a corneal epithelial phenotype (Fig. 5B).
Following the stem cell transplantation procedure, the
patient was treated topically with eye drops as outlined in
Supporting Information data, Annex 3. No systemic immuno-
suppression was needed since all transplanted tissue was auto-
logous in nature (except for the HAM, which is generally
accepted as non-immunogenic). Within 1 week of surgery, the
patient’s left eye was significantly less red and more comfort-
able (Supporting Information Fig. 10). Initially, there was
mild peripheral vascularization of the superficial HAM, but
this HAM melted within the first four weeks to reveal a
healthy avascular layer of transplanted ex vivo expanded tis-
sue, which formed a stable epithelium with no epithelial
defect. This was associated with an absence of ocular surface
inflammation and a comfortable eye over the next 12 months
with no side effects. We then elected to proceed to central
full thickness corneal graft to replace the previously scarred
stroma. Such a graft would be expected to succeed since it
would now be reepithelialized by healthy host limbal epithe-
lium. The patient therefore underwent a left HLA-matched
penetrating keratoplasty 1 year after the stem cell graft (data
not shown). The removal of the central corneal button gave
us a unique opportunity to perform detailed analysis of the
corneal epithelium, which must have been generated from the
previously transplanted autologous LSCs as our clinical data
had shown a complete absence of normal corneal epithelium
on the ocular surface for over 10 years. The histology and im-
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605
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munohistochemical analysis showed that a multilayered strati-
fied nonkeratinizing epithelium was formed on a basement
membrane (Supporting Information Fig. 11). Periodic acid-
Schiff (PAS) staining revealed the complete absence of glyco-
gen containing goblet cells found in the previously conjuncti-
valized corneal surface. The p63 staining demonstrated the
presence of proliferating LSC-like cells in the basal epithe-
lium that were not present in the most superficial layers. In
addition, all the cells of the corneal epithelium now expressed
the corneal specific marker, CK3. TEM revealed that the
ultrastructure of the generated epithelium shows all the fea-
tures of a normal central corneal epithelium (Supporting In-
formation Fig. 12). It shows in detail a uniform stratified epi-
thelium. The basal layer of cells are columnar and rest on a
basement membrane and are attached to it by means of hemi-
desmosomes. The epithelial cells are attached to one another
by desmosomes. The most superficial layer of squamous cells
is differentiated to show multiple microplicae.
The long-term follow-up data of all these outcome meas-
ures is shown in Figure 6A and 6B. In summary, we can
conclude for the first case study that transplantation of ex
vivo expanded autologous limbal epithelium using an
explant technique on intact HAM cultured with a non-human
animal cell free, GMP compliant system, leads to successful
and long-term (at least 2 years) reversal of LSCD. This is
shown by the successful objective outcome measures includ-
ing normal impression cytology (CK19 negative) and ab-
sence of epithelial defect following transplantation. In addi-
tion there is improvement of Snellen’s visual acuity, reversal
of corneal vascularization, and decreased corneal opacity.
There is also a significant decrease in pain and visual
impairment scores.
606 Successful Stem Cell Therapy for Unilateral LSCD

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Figure 6. Summary of outcomes of ex vivo expanded limbal epithelium transplantation for Patient 1. (A): Our treatment protocol produces re-
versal of LSCD by all measures including impression cytology, reversal of epithelial defects, corneal vascularization, corneal opacity, and Snellen
vision. Corresponding subjective scores show a corresponding reduction in pain and visual impairment scores. (B): Microphotographs showing
the patient’s affected eye before and after the limbal and corneal transplant. Abbreviations: LSCD, limbal stem cell deficiency.

Summary of Clinical Results from Transplantation
of Ex Vivo Expanded Limbal Epithelial Cells in
Eight Patients with Unilateral Total LSCD
Eight patients, including the above first case (that fulfilled all
the inclusion and exclusion criteria; Supporting Information
data, Annex 1) were included in this study (for patient demo-
graphics, refer to Supporting Information Table 1). All
patients underwent limbal biopsy of their healthy fellow eye
using our standard biopsy protocol. Excellent outgrowths
were obtained for each patient with initial outgrowths noted
by day 2-3. The growth of the explant culture was marked at
each feed day and the results of the growth are shown in Sup-
porting Information Figure 13. The patients have been fol-
lowed up for an average of 19 months following LSC treat-
ment (range 12-30 months). The primary subjective measure
was reduction in ocular discomfort. Improvement in patient’s
vision was a secondary subjective outcome measure as the
majority of patients had significant corneal stromal scarring,
secondary to the original disease, and recurrent inflammation
caused by LSCD. The subjective outcomes are shown in Fig-
ure 7A and 7B. These results clearly show that, in all cases,
there has been a significant improvement in both subjective
outcomes. The pain scores reduced from 7.25 (SD ¼ 1.28)
pretreatment to 0.75 (SD ¼ 0.89) at the end of the follow-up
period, which is highly significant (p < .0001, Student t-test).
The visual impairment scores were also reduced from 7.63
(SD ¼ 1.30) pre-treatment to 3.00 (SD ¼ 2.45), which was
also highly significant (p ¼ .00033, Student t-test).
A summary of primary and secondary objective outcome
measures is also shown in Figure 7C. From this, it is clear
that 100% of patients had successful reversal of their LSCD
as shown by change of their central corneal epithelium
impression cytology from a conjunctival phenotype preopera-
tively to a corneal phenotype postoperatively as well as suc-
cessful re-epithelialization following transplantation of ex
vivo expanded LSC with no significant epithelial defects in
any patient at the end of the study period. There was also a
significant improvement in secondary objective measures such
as LogMAR visual acuity (Figure 7D), reversal of central cor-
neal vascularization, and reduction of corneal capacity.
By the end of the follow-up period, satisfactory ocular sur-
face reconstruction was obtained in all eyes (100%), as confirmed
by clinical examination and impression cytology. However, three
of the eight eyes developed localized conjunctival invasion of the
cornea (less than 3 clock hours) within the first 6-12 months. All
three patients underwent sector epitheliectomy where the tongue
of conjunctival in-growth was scraped off the corneal periphery
combined with HAM transplant as a biological bandage (stroma
side down), allowing surrounding LSCs to reform the limbal bar-
rier. This was successful in reversing the localized conjunctivali-
zation without the need for additional LSC transplant.
Kolli, Ahmad, Lako et al. 607

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Figure 7. Graphs to show subjective and objective outcomes of ex vivo expanded limbal stem cell (LSC) transplantation. (A): Pain scores
shown above, and visual impairment scores shown below. (B): Improvement in subjective outcomes as a result of LSC transplantation. (C):
Objective outcomes from preoperative values (Preop) to postoperative values at the end of the follow-up period (Postop). For Patient 1 who
underwent a penetrating corneal transplant 1 year after LSC transplant, the precorneal graft values of visual acuity and corneal opacity were used
to prevent bias from the second procedure. (D): Changes in LogMAR Visual Acuity (this is a linear scale allowing statistical comparison of
vision where lower values correspond to better visual acuity) following ex vivo expanded limbal epithelium for the treatment of total limbal stem
cell deficiency.

J2-3T3 fibroblasts with FCS supplemented media [14]. Since

DISCUSSION
The first use of ex vivo expanded LSCs for the treatment of
LSCD in human subjects was described by Pellegrini and co-
workers, who used a culture system of LSCs grown on mouse
www.StemCells.com
then, several studies have been reported on the transplantation
of ex vivo cultured LSCs for the treatment of LSCD [14, 25–
28, 39–51]. It is difficult to draw conclusions from existing
studies because of the lack of standardization in terms of
patient selection (such as total and partial LSCD used in the
608
Figure 7.
same study), cause of LSCD (acquired and congenital), unilat-
eral and bilateral cases, source of initial tissue (allo- and
auto-graft transplants in the same study), methods of ex vivo
expansion (explant or single cell; HAM or 3T3 fibroblast co-
culture or both), the surgical management (method of superfi-
cial keratectomy, the use of a second HAM as a bandage,
contact lens protection, or both) and postoperative manage-
ment (use of HAS or not). These factors represent a major
deficiency in this field of LSC therapy and also a major obsta-
cle to interpret the results. We have aimed to improve this by
describing a novel, fully validated non-human animal cell free
system under GMP conditions with well defined inclusion and
exclusion criteria and specific subjective and objective out-
come measures.
Our system of ex vivo expansion uses a very specific and
well defined technique of intact HAM stretched onto glass
coverslips together with an explant technique. The culture me-
dium used was modified epithelial growth medium where the
FCS was replaced by HAS. This combination has been used
successfully over the last few years in our laboratory studies
Successful Stem Cell Therapy for Unilateral LSCD
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(Continued)
where we have shown that LSCs can grow from the limbal
explant onto the HAM [38]. The GMP culture process used
shows categorically that a system of ex vivo expansion of
very small amounts of limbal tissue can be used to produce
large sheets of cells that can successfully regenerate the cor-
neal epithelium in patients with total LSCD. Analysis of the
expanded sheets at the time of surgery shows that they con-
tain an epithelium with a basal layer of cells that have a
LSC-like morphologic appearance and express a signature of
putative LSC markers. These findings are in agreement with
our previous detailed laboratory analysis of ex vivo expanded
cultures [2, 38, 52] using explant cultures on intact HAM.
To ensure the subsequent success of transplanted cells, the
host environment must be made conducive to cell survival.
Thus, we took a great deal of effort to remove all the abnor-
mal conjunctival epithelium on the diseased eye and minimize
bleeding. We also placed an additional HAM on top of the
cell culture, which we feel not only would protect the trans-
planted cells from physical trauma but may produce addi-
tional LSC niche cues that prolong the life span and
Kolli, Ahmad, Lako et al.
maintenance of clonogenicity of epithelial progenitor cells
[29] while inhibiting inflammation [52] and vascularization
[52] and promoting corneal epithelialization [53]. At the
time of surgery, the abnormal conjunctival epithelium was
removed and subsequently analyzed both histologically and
with immunohistochemistry to provide tissue diagnostic proof
that no normal corneal epithelium existed in our patients
preoperatively.
Postoperatively, in addition to topical steroid and antibiot-
ics, we also treated all patients with up to a one-year course of
autologous serum, which has been shown to have significant
epitheliotropic effects [35] and to be a useful adjunct to ocular
surface reconstruction [55]. Close postoperative follow-up of
the patient is vital, particularly in the first 12 months. Rapid
and stable epithelialization took place in all our patients so that
there were no epithelial defects within a few days post-trans-
plantation. If there was a localized area of active peripheral
vascularization representing an area of incoming conjunctival
epithelium through a defect in the limbal barrier (as was the
case in three patients), we used the technique of sequential sec-
tor conjunctival epitheliectomy described by Dua et al. [56] to
excise the small incoming tongue of conjunctival epithelium
and allow the transplanted LSCs to restore the limbal barrier
(which proved successful in each case).
Successful surgical treatment of LSCD was proven for each
patient in this study both from reversal of the clinical signs, in
particular the resurfacing of the corneal surface with a stable
epithelium, and also by the conversion of impression cytology
specimens of the central cornea epithelium from a conjunctival
phenotype preoperatively to a corneal phenotype postopera-
tively. There was a marked improvement in subjective symp-
toms in all patients, many of whom had suffered years of per-
sistent ocular pain and significant visual reduction. Interestingly,
those patients who had a fairly recent diagnosis of total LSCD
had marked improvement in objective visual acuity since the
main issue of conjunctivalization had been reversed. However,
for those with a several-year history of LCSD, the visual
improvement was not as marked since persistent epithelial
breakdown and inflammation combined with recurrent infection
led to marked corneal stromal scarring. Although these patients
had a stable epithelium after LSC transplantation, they would
require a corneal transplant to restore vision.
In conclusion, we have succeeded in using a non-human
animal product free GMP-compliant autologous LSC ex vivo
expansion technique to successfully reverse LSCD within a
controlled population and showed 100% success in predefined
subjective and objective outcome measures. However, there
are still a few unanswered questions. Firstly, the initial
patients transplanted using these techniques have only com-
pleted just over 2 years postoperative follow-up in this study
and we are still unsure about very long-term outcomes, which
are pending, although results so far are very encouraging.
Although a healthy epithelium can be produced using this
technique in an eye with no observable pre-existing LSCs,
there are as yet no direct ways of accurately identifying LSCs
in vitro or in vivo due to the absence of a specific marker.
Although HAM appears to be a very effective means of cul-
turing LSCs, it is, however, a biological substrate that is
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spective. Bull World Health Organ 2001;79:214– 221.
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www.StemCells.com
609
impossible to standardize in terms of its structure (e.g., thick-
ness), physiological properties, and handling, and there is
scope for finding reliable and consistent alternatives for a cul-
ture substrate. A previous study has used a fibrin support for
transplantation of ex vivo cultured LSCs in combination with
FCS and 3T3, although their success rate was slightly lower
than ours (success was achieved in 14 out of 18 patients). It
remains to be investigated whether a combination of the fibrin
support with our culture conditions will provide a fully stand-
ardized system with equal efficiency in treatment of patients
with LSCD [57].
The role of the niche in maintaining the optimal environ-
ment for the protection and maintenance of LSCs is becoming
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better understood. In addition, in our patient group who have
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niques to directly identify LSCs in vivo. We envisage a time
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ACKNOWLEDGMENTS
We would like to thank all those in the Department of Oph-
thalmology at the Royal Victoria Infirmary [Newcastle upon
Tyne NHS Hospitals Foundation Trust] who enabled the pro-
gression of this work to the clinical stage. In addition we are
very grateful to Prof. Anne Dickinson, Dr. Andy Gennery,
Janice Dunn, and Ken Brigham for help in the good manufac-
turing practice laboratory. Without their help, the transplants
would not have been possible. We are also grateful to Dr.
Hardeep Mudhar [Sheffield Teaching Hospitals NHS Foun-
dation Trust] for his input in providing independent confirma-
tion of all histological specimens. We would also like to
thank the Newcastle Healthcare Charity, Life Knowledge
Park, One North East Developmental Agency, U.K. NIHR
Biomedical Research Centre for Ageing and Age-related Dis-
ease, and the Newcastle upon Tyne NHS Hospitals Trust for
their financial support for this research and the United King-
dom Eye Banks for providing access to human limbal tissue
donated for research. Finally, we would like to thank the
donors of limbal tissue for donating their tissue for research.
Because of their generosity, patients with limbal stem cell
deficiency will be able to have their sight restored and lead
much more comfortable lives.
DISCLOSURE OF POTENTIAL
CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.
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